Project Summary/Abstract Gene expression, together with genome engineering, technology are the cornerstones of the biotechnology revolution. While much advances have been made, most gene expression control systems were designed to control a single gene. However, many biological processes, such as developmental and cancer progression, are driven by the simultaneous changes of expression for multiple genes in a sequentially and spatially controlled fashion. As our genome editing capabilities rapidly progress, the development of gene expression control technology has fallen behind. Advancement in gene expression technology that enables multiplexed spatiotemporal control is therefore urgently needed to directly interrogate complex biological processes and to engineer novel phenotype for biotechnological applications. Site-specific DNA recombinase (SSR) (e.g., Cre and Flp) has become one of the most powerful gene regulation tools in mammalian cells. We and others have shown that recombinases are uniquely capable of creating exceptionally complex logic circuits with high robustness. As such, recombinase represents an ideal foundation for engineering advanced gene expression control systems. Most of the recombinase-based expression technologies were designed using one enzyme, Cre, which affords limited functionality concerning the number of genes that can be regulated independently in a spatiotemporal manner. To develop the next generation of recombinase-based gene expression technology, robust orthogonal inducible recombinases are necessary. Leveraging our vast experience in engineering inducible recombinases and recombinase-based circuit, we will develop a suite of advanced recombinase-based tools that show simultaneous, sequential, and/or spatially controlled tuning of the expression of multiple chosen genes. In particular, we will Aim 1: Develop orthogonal small molecule inducible recombinases for simultaneous control of multiple gene expressions independently in the same cell. Aim 2: Develop multichormatic light inducible gene switches for spatial control of gene expression Aim 3: Develop cascade circuits for sequential control of gene expression We will validate our system in human and mouse cells to ensure broad applicability. We will develop metric and datasheet, a database for DNA repository to facilitate adoption and sharing. My group is uniquely capable of accomplishing this proposed work because of our published expertise in 1) DNA recombinases and 2) genetic circuit designs. Success from this proposed work will dramatically increase our capability to control gene expression in mammalian cells with enhanced spatiotemporal precision.